The consideration of individual parameters of the cellular energy metabolism for the evaluation of the potential of metastasis of malignant tumors has often been tested, however without transferring test results or test procedures into clinical every day life (Novartis Foundation symposium 240, 251 (2001)). The concentration of substrates and products of the energy metabolism such as glucose, glutamine, ketone bodies, lactate and ATP have been determined in cell lines in xenograft tumors, in cryo tissue samples of tumor patients and also in vivo in larger vessels supplying a tumor (Cancer research 62, 6674 (Nov. 15, 2002)). These determinations are based on methods which are generally known in the analysis of metabolism such as enzyme activity tests, bioluminescence tests, NMR or mass spectrometry. Presently, there is no clinical test in which all essential energy metabolism pathways in tumor tissue are tested to give evidence about an individual prognosis of a tumor or about the chemosensitivity of a tumor.
Several published experimental approaches for an application of enzyme activity tests of energy metabolism enzymes have not been considered in clinical therapy of human malignant tumors in spite of the fact that these enzyme activities are different in healthy and tumor tissue. Determination methods are necessary which allow a quick decision on a therapy and which allow a reliable prognosis of the tumor development after tumor diagnosis. The presently available methods for analysis of the energy metabolism of malignant tumors do not fulfill the aforementioned requirements.
It would be highly beneficial if such methods that are performed at tissue samples—herein the term “tissue samples” comprises solid tissue and single cells from liquid material (i.e. blood)—would both provide results that are identical to those in vivo of a patient and allow an interindividual cross-comparison of the measured enzyme activities. This is not possible with an analysis of formalin-fixed tissue, cell lines, spheroids and xenograft tumors. A representation of the energy metabolism in vivo could be a cryo tissue sample taken from biopsy material. Such samples allow the determination of metabolites; the determination of activities of enzymes that are located in cell compartments or are part of multienzyme complexes is limited. However, measurements in cryo tissue have the disadvantage that individual differences of patients in energy and substrate supply prior to taking the biopsy material from a patient and the changes in tumor metabolism induced by the surgeon during the biopsy have an influence on the enzyme activity. Such circumstances do not allow standardization and interindividual cross-comparison of measured metabolic data. Already the duration of the biopsy, the kind and the dosage of a narcotic during biopsy or drug administration may distinctly influence energy metabolism in a cell (Resuscitation 19, 159 (April 1990).
Other methods in which the expression of enzymes of the energy metabolism in patient tissue is determined via immunohistochemistry or bioluminescence are semiquantitative and do not indicate the actual activity of the enzymes. Nature Chemical Biology 1 130 (August 2005) describes the so-called “activity-based-proteome-profiling” or its further developed application (click-chemistry) that show active enzymes in homogenates and living tissue; however, they are semiquantitative, very time consumptive and costly.
Up to now, none of the aforementioned methods or combination of such methods allows the determination or representation of the complete energy metabolism of living tumor tissue. In particular, the activity of key enzymes involved in the metabolism of all known relevant cellular energy substrates has not yet been considered and determined for a prognosis of tumor progression or for a therapy recommendation.